Discrimination of <i>Bacillus subtilis</i> from Other <i>Bacillus</i> Species Using Specific Oligonucleotide Primers for the Pyruvate Carboxylase and Shikimate Dehydrogenase Genes

Abstract

<i>Bacillus subtilis</i> is a useful bacterium in the food industry with applications as a starter strain for fermented food and as a probiotic. However, it is difficult to discriminate <i>B. subtilis</i> from other <i>Bacillus</i> species because of high phenotypic and genetic similarity. In this study, we employed five previously constructed multilocus sequence typing (MLST) methods for the discrimination of <i>B. subtilis</i> from other <i>Bacillus</i> species and all five MLST assays clearly distinguished <i>B. subtilis</i>. Additionally, the 17 housekeeping genes used in the five MLST assays also clearly distinguished <i>B. subtilis</i>. The pyruvate carboxylase (<i>pyrA</i>) and shikimate dehydrogenase (<i>aroE</i>) genes were selected for the discrimination of <i>B. subtilis</i> because of their high number of polymorphic sites and the fact that they displayed the lowest homology among the 17 housekeeping genes. Specific primer sets for the <i>pyrA</i> and <i>aroE</i> genes were designed and PCR products were specifically amplified from <i>B. subtilis</i>, demonstrating the high specificity of the two housekeeping genes for <i>B. subtilis</i>. This species-specific PCR method provides a quick, simple, powerful, and reliable alternative to conventional methods in the detection and identification of <i>B. subtilis</i>.